Apparatus and method for performing mass spectroscopy

ABSTRACT

Embodiments of the present invention are directed to apparatus and methods for performing mass spectrometry. Data pair information is subjected to an ion audit process in which data pair information that relates to scored compounds is subtracted from the data pair information. The depleted information more readily reveals data pair information for compounds present with smaller signals.

This application is a continuation of application Ser. No. 12/301,469,which is the National Stage of International Application No.PCT/US2007/069657, filed on May 24, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/808,815, filed May 26, 2006. Theentire contents of all of these applications are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to apparatus and methods for performing massspectroscopy and, in particular, identifying compounds in a mixture bymass and chromatographic retention times.

BACKGROUND OF THE INVENTION

Mass spectrometry is a technique for determining the mass of a compound.Mass spectrometers are instruments that produce a mass to charge signalthat can be processed or interpreted to suggest a mass of a compound.Mass spectrometers place a charge on molecule and accelerate themolecule to a detector. The detector produces a signal relating to themass of the molecule and the charge carried on the molecule. Massspectrometry is used to identify proteins and other compounds ofbiological origination.

Chromatography is a technique for separating compounds held in solution.Compounds held in a solution will exhibit different affinity for a solidmedium in contact with the solution. As a solution flows past or throughan immobile medium, the compounds separate from each other.

As used herein, the term “retention time” refers to the time a compoundtakes to exit from a column or cartridge or other separation devicecontaining a solid separation medium.

As used herein, the term “sample” refers to the material being analyzed.Samples of biological origin are often complex mixtures comprisingproteins, precursors of such proteins, fragments of proteins andreaction products of proteins and other compounds. Proteins are capableof assuming different charge states and being fragmented. Often, theanalysis of the sample may include an enzymatic digestion to cleave thesample at one or more known sites. These digestions may go to completionor leave incomplete digestion products. In a typical analysis, a samplecomprising one or more proteins is placed in a vial and subjected toenzymatic digestion. The digested sample is passed to thechromatographic apparatus and separated into the products of thedigestion.

It is common to combine chromatography with mass spectrometry.Chromatography is used to separate the compounds of a sample, whichcompounds are placed into a mass spectrometer. The mass spectrometerproduces a mass to charge signal that can be related to a retentiontime.

The data produced in such a combination is complex and difficult toanalyze. Methods and apparatus to analyze such chromatographic massspectrometry data with greater specificity and fewer falseidentifications are desired.

SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to methods andapparatus for analyzing samples. One embodiment of the present inventionfeatures an apparatus identifying compounds in a mixture by mass andchromatographic retention times. The apparatus comprises computerprocessing means placed in signal communication with a chromatographicapparatus and a mass spectrometer for receiving one or more signals fromthe chromatographic apparatus relating to retention time and one or moresignals from a mass spectrometer relating to mass. The chromatographicapparatus and the mass spectrometer are in fluid communication to allowthe chromatographic apparatus to receive samples and pass such samplesto the mass spectrometer. The computer processing means correlates eachmass signal to a retention time signal to form a data pair and data pairinformation for a plurality of data pairs, storing the data pairinformation, and sorting the data pair information into groups definedby a range of retention times into bins. The computer processing meanshas data base means comprising mass and retention time information forknown compounds. The computer processing means initiating an ion auditprocess having a first pass.

The first pass of the ion audit process comprises the step of specifyingmatching criteria for data pair information present in a bin or aplurality of bins, potentially representing a compound, to form matchparameters and identifying a group of data pairs corresponding to thematch parameters, said data pairs corresponding to the match parametersforming a matched set. Next, the matched set is compared to the database means and identifying a compound corresponding to at least one ofthe match parameters and assigning an identification to the data pairsof the matched set to form an scored compound. The data pairs of thematched set is subtracted from the bins to form one or more depletedbins. This process is repeated with the data pair information held inthe one or more bins and depleted bins, until the ion audit processassociates all data pair information with compounds, fragments ofcompounds and reaction products of compounds, or the match criteria forremaining compounds, fragments of compounds and reaction products ofcompounds do not correspond to information in the data base, or a userdefined number of repetitions.

Preferably, the ion audit process focuses on the dominant signals, theaudit clears such signals from the data pair information to allowidentification of the next dominant signal group. Thus, as the auditprogresses, the lower intensity signals are revealed with ever greaterclarity.

The term “computer processing means” is used to denote software andfirmware for use by computers, programmed computer processing units(CPUs), personal computers, servers, mainframe computers, computers andCPUs integrated with chromatographic apparatus or mass spectrometers orother analytical instrumentation.

The term “communication”, with reference to fluids, means plumbedtogether. The term “communication” with reference to signals, meanswired, or optically linked, or radio signal linked as in wirelesscommunication so as to receive and/or emit signals.

The term “correlates” is used to denote linking or combining. Forexample, combining a retention time with a mass to charge signal meansthat the mass to charge signal which was produced by a ion with adefined retention time is held together a pair of values, a data pair.The term “bin” or “bins” is used to denote a group of such data pairswith common or similar features. The present application forms bins witha range of retention times. That is, the total time of a separation isdivided into a plurality of time periods.

The term “data base means” refers to an archive of information. Thisdata base means can be developed by a user from data developed andstored in one or more computers, a commercially available data baseavailable through network systems, such as the internet, or availablethrough access through storage devices and the like.

The term “matching criteria” refers to values that fall within a definedrange. This matching criteria may be a cluster or grouping of values.The term “parameters” is used to denote the values that are used tocompare values found in the data pairs or in the data base. Data pairsthat comport with the parameters are grouped in matched sets.

A “scored compound” is the putative identity of the compound that isrepresented by the set of data pairs. The identity may have differentdegrees of confidence, which upon further analysis of the data mayincrease or decrease.

Preferably, the ion audit process comprises specifying matching criteriafor data pair information present in a bin, depleted bin or a pluralityof bins or depleted bins, potentially representing one or more fragmentsof the scored compound, to form fragment match parameters. Next, a groupof data pairs corresponding to the fragment match parameters areidentified to form a group of data pairs forming a fragment associatedmatched set. The fragment associated matched set is subtracted from thebins and depleted bins. The further depleted bins have fewer data pairsallowing other groups of data pairs to be recognized with greaterclarity. The data pairs which represent fragments of the scored compoundare used to increase confidence in the compound identification.

Preferably, the ion audit comprises specifying matching criteria fordata pair information present in a bin or depleted bin or a plurality ofbins or depleted bins, potentially representing one or more precursorsof the scored compound, to form precursor match parameters. Next, agroup of data pairs corresponding to the precursor match parameters areidentified, to form a group of data pairs forming a precursor associatedmatched set. The precursor associated matched set is subtracted from thebins and depleted bins. The further depleted bins have fewer data pairsallowing other groups of data pairs to be recognized with greaterclarity. The data pairs which represent precursors of the scoredcompound are used to increase confidence in the compound identification.

The term “precursor” is used to denote both an incomplete digestionproduct of a protein or a larger protein from which the scored proteinis made or smaller parts of a protein which are condensed or foldertogether to form a protein. For example, in vivo, proteins aresynthesized with added amino acids or peptide units that may function inestablishing the tertiary structure, that is, how the protein folds ontoitself, these added amino acids or peptide units may be lost or removedto produce the active protein.

Preferably, the ion audit comprises specifying matching criteria fordata pair information present in a bin or depleted bin or a plurality ofbins or depleted bins, potentially representing reaction products of thescored compound, to form reaction product match parameters. Next, datapairs which correspond to the reaction product match parameters areidentified to form a group of data pairs forming a reaction productassociated matched set. The reaction product associated matched set issubtracted from the bins and depleted bins. Again, the further depletedbins have fewer data pairs allowing other groups of data pairs to berecognized with greater clarity. The data pairs which represent reactionproducts of the scored compound are used to increase confidence in thecompound identification.

The apparatus can perform the ion audit in a single pass of all bins ora subset or a single bin. However, it is preferable to apply thesubtraction to a plurality of bins such that data pairs associated witha putative compound are removed and other details seen.

Preferably, the ion audit process includes a second pass, wherein for ascored compound, at least one selected from the group consisting ofanticipated fragments, anticipated precursors and anticipated reactionproducts thereof, are identified from the data base for the scoredcompound. Matching criteria for data pair information present in one ormore bins to form are specified defining second pass match parameters tosaid selected anticipated fragment, anticipated precursor andanticipated reaction product thereof. Data pairs which correspond tosaid second pass match parameters are identified to form a group of datapairs forming a second pass matched set. The second pass matched set issubtracted from said bins and depleted bins.

Again, the further depleted bins have fewer data pairs allowing othergroups of data pairs to be recognized with greater clarity. The datapairs which represent anticipated fragments, anticipated precursors andanticipated reaction products of the scored compound are used toincrease confidence in the compound identification.

The term “anticipated fragment” is used in the sense of incompletedigestions of a protein, or a fragment that is normally encountered inmass spectrometers having a fragmentation chamber. In the event thechromatographic apparatus receives a digest of a sample, incompletedigestion processes can be anticipated.

In the event the mass spectrometer has a fragmentation chamber, thecompounds can be expected to form fragments. A preferred massspectrometer has a fragmentation chamber and has a low energy mode inwhich fragments are not formed and a high energy mode in which fragmentsare made. A preferred mass spectrometer can switch from high to lowenergy mode within a time interval smaller than the interval associatedwith the bins.

The term “anticipated reaction product” is used to denote commonmethylated, oxidized phosphorylated and glycosylated derivatives of thecompounds and the fragments, and precursors of the compounds.Anticipated precursors are molecules from which the compounds are madewhich would be expected to be present in a sample.

Preferably, the apparatus further comprises or is used with data outputmeans for displaying the compounds identified in the ion audit process.The data output means may comprise printers, video output devices, suchas video monitors, screen displays, projectors.

The apparatus further comprises or is used with chromatograph apparatusfor receiving a sample and separating said sample into compounds byretention time. The chromatographic apparatus is in signal communicationwith computer processing means to produce a signal from which retentiontime can be determined.

The apparatus further comprises or is used with a mass spectrometer forreceiving compounds from the chromatographic apparatus and producing asignal corresponding to the mass of the compound. And in the event saidcompound forms one or more fragments, a signal corresponding to afragment, and in the event the compound reacts to form a reactionproduct, a signal corresponding to said reaction product, and in theevent the compound is present as a precursor, a signal corresponding tothe precursor, the mass spectrometer in signal communication with saidcomputer processing means to produce a mass signal relating to the massof a compound, fragment, reaction product or precursor;

A further embodiment of the present invention is directed to a method ofidentifying a compound with chromatographic apparatus and massspectrometry apparatus. The chromatographic apparatus receives a sampleand separates the sample into compounds by retention time. And, thechromatographic apparatus produce a signal from which retention time canbe determined. The mass spectrometer receives one or more compounds fromthe chromatographic apparatus and produces a signal corresponding to themass of the compound. And, in the event the compound forms one or morefragments, a signal corresponding to a fragment, and in the event thecompound reacts to form a reaction product, a signal corresponding tothe reaction product, and in the event the compound is present as aprecursor, a signal corresponding to the precursor. The methodcomprising an ion audit process having the steps of correlating eachmass signal to a retention time signal to form a data pair and data pairinformation for a plurality of data pairs, storing the data pairinformation, and sorting said data pair information into groups definedby a range of retention times into bins. The method further comprisesthe step of specifying matching criteria for data pair informationpresent in a bin or a plurality of bins, potentially representing acompound, to form match parameters. And, the method comprises the stepof identifying a group of data pairs corresponding to the matchparameters, the data pairs corresponding to the match parameters forminga matched set. Next, the matched set are compared with data base meansand a compound is identified that corresponds to at least one of thematch parameters to assign an identification to the data pairs of thematched set to form an scored compound. The data pair information ofsaid matched set are subtracted from the bins to form one or moredepleted bins. The ion audit of the present invention repeats the stepsof specifying matching criteria through the step of subtracting datapair information from the bins and depleted, until all data pairinformation is associated with compounds, fragments of compounds andreaction products of compounds, or the match criteria for remainingcompounds, fragments of compounds and reaction products of compounds donot correspond to information in the data base, or a user defined numberof repetitions.

Preferably, the method comprises the step of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing one or more fragments of the scoredcompound, to form fragment match parameters. Next, a group of data pairscorresponding to the fragment match parameters are identified to form agroup of data pairs forming a fragment associated matched set. The datapair information of fragmented associated matched sets is subtractedfrom the bins and depleted bins. The data pairs which representfragments of the scored compound are used to increase confidence in thecompound identification. Removal of the data pair information allowsother features of the data to be seen.

Preferably, the method comprises the step of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing one or more precursors of the scoredcompound, to form precursor match parameters. Next, a group of datapairs corresponding to said precursor match parameters are identified toform a group of data pairs forming a precursor associated matched set.The data pair information of the precursor associated matched set issubtracted from the bins and depleted bins. The data pairs whichrepresent precursors of the scored compound are used to increaseconfidence in the compound identification. Removal of the data pairinformation allows other features of the data to be seen.

Preferably, the method comprises the steps of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing a reaction products of the scoredcompound, to form reaction product match parameters. Data pairs whichcorrespond to said reaction product match parameters are identified toform a group of data pairs forming a reaction product associated matchedset. The data pair information of the reaction product associatedmatched set is subtracted from the bins and depleted bins. The datapairs which represent reaction products of the scored compound are usedto increase confidence in the compound identification. Removal of thedata pair information allows other features of the data to be seen.

The method of the present invention can be performed in a single firstpass or in multiple passes. Preferably, the ion audit process includes asecond pass, wherein for a scored compound at least one selected fromthe group consisting of anticipated fragments, anticipated precursorsand anticipated reaction products thereof is chosen for a second pass.The second pass follows a first pass or a first series of passes inwhich the specifying criteria remains substantially unchanged.

These and other features and advantages of the present invention will beapparent to those skilled in the art from viewing the Figures andreading the detailed description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of an apparatus embodying features of the presentinvention;

FIG. 2 a is a spectrum of binned data pairs without depleting the bins;

FIG. 2 b is a spectrum of depleted binned data pairs;

FIG. 3 is a flow diagram of an apparatus and method embodying featuresof the present invention; and

FIGS. 4 a and 4 b are a flow diagram of an apparatus and methodembodying features of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to theFigures with respect to methods and apparatus for analyzing samples. Thedescriptions in this regard will highlight the use of the invention forchemical analysis of proteins and peptides. However, those skilled inthe art will recognize that embodiments of the present invention havebroader application for the analysis of other compounds or compositions,polymers, and biopolymers including without limitation, nucleic acids,carbohydrates, and synthetic polymers.

An apparatus embodying features of the present invention, generallydesignated by the numeral 11, is depicted in FIG. 1. The apparatus isused to identify compounds in a mixture by mass and chromatographicretention times. The apparatus comprises the following major elements;computer processing means 15, a chromatographic apparatus 17, and a massspectrometer 19.

As depicted, the computer processing means 15 suggests a computer orCPU. However, the computer processing means 15 may comprise software orfirmware, loaded on a computer, CPU or server mainframe computer or CPUintegrated with other analytical equipment, including by way of example,the chromatographic apparatus 17 and mass spectrometer 19. Software iscommonly programmed to the memory of a computer by means of a computerreadable device or disk or networks having access to the computerprogram. Once programmed, the software is considered firmware. Computersand CPUs are known in the art and sold by numerous vendors. IBM formatcomputers are sold by Dell Computer Corporation and others. Apple formatcomputers are sold by Apple Corporation (California, USA). By way ofexample, computer processing means 15 may comprise s computer programsintegrated into larger software programs for managing the operation ofanalytical instruments, data management and data processing. Such largersoftware programs are sold by several vendors under the trademarksMILLENNIUM®, EMPOWER™ (Waters Corporation, Milford, Mass., USA),CHEMSTATION™ (Agilent Corporation, Palo Alto, Calif., USA), MASSLINKS®(Micromass UK Ltd, Manchester, England) and others.

Computer processing means 15 is in signal communication withchromatographic apparatus 17 and a mass spectrometer 19 as depicted bylines 23 a and 23 b. Those skilled in the art will recognize that theline 23 a and line 23 b can be replaced with wireless communicationdevices and routers. Line 23 a and line 23 b can be wires for electricalmagnetic signals or represent optical communication means such as fiberoptic cable.

Computer processing means 15 is in signal communication withchromatographic apparatus 17 and mass spectrometer 19. Chromatographicapparatus 17 comprises any number of chromatographic apparatus includingby way of example without limitation , liquid and gas chromatographicinstruments. Chromatographic apparatus typically have a pump forpropelling a fluid [not shown] and conduits for carrying the fluid.Chromatographic instruments receive sample from a sample manager orsample injection device [not shown]. As depicted, the chromatographicapparatus 17 is in fluid communication with a sample held in a vial 25by means of a conduit 27. This sample, when proteins and peptides areanticipated, held in vial 25, is subjected to digestion means such asenzymes and the like. This causes the proteins and peptides to form oneor more digestion products.

Chromatographic instruments receive and place a sample into a solventflowing in a conduit [not shown]. The solvent is received in aseparation device [not shown] such as a capillary, column or cartridge.Columns and cartridges are commonly packed or contain a separation mediawhich interacts with the sample compounds in the solvent. Thesecompounds will exhibit different affinity for the separation media andwill be retained and separate. The period of time the compounds areretained is known as the retention time.

Chromatographic apparatus 17 produces one or more signals which arecommunicated to the computer processing means relating to retentiontime. Chromatographic apparatus of the type depicted are available fromseveral vendors under the tradenames ALLIANCE® and ACQUITY ® from WatersCorporation and 1100™ from Agilent GmbH of (Germany).

As depicted, chromatographic apparatus 17 is in fluid communication withmass spectrometer 19 by means of a conduit 29. However, fluidcommunication, in this sense, does not require that the massspectrometer 19 receives sample from the chromatographic apparatus 17 bymeans of a conduit 29. The mass spectrometer 19 may receive samplerelated to retention time by deposition on a solid surface in the natureof MALDI or DIOS process.

The mass spectrometer 19 produces a signal relating to mass, and, moreprecisely relating to the mass to charge ratio of a compound. Massspectrometer 19 is illustrated as a triple quadrupole mass spectrometerhaving a first mass selection chamber 31, a fragmentation chamber 33 anda mass detection chamber 35. The first mass detection chamber 31 selectsan ion or group of ions by mass to charge ratio. The ions may be allowedto pass through the fragmentation chamber 35 or subjected to energywhich causes the ions to from fragments. The ions and ion fragments arepassed to the mass detection chamber 35.

Mass spectrometers such as mass spectrometer 19 are available fromseveral vendors including Waters Corporation (Milford, Mass., USA),Thermo Finnigan (Waltham, Mass., USA), Agilent Corporation (Palo Alto,Calif., USA) and others.

The computer processing means 15 correlates each mass signal to aretention time signal to form a data pair or group. By way of example,the data may be organized as set forth in Table 1 below:

TABLE 1 MH+ Intensity Retention time Charge State 484.7397 110 43.556 1

The data pair information for a plurality of data pairs is stored. Thedata pair information may comprise a thousand fold lines of the type setforth above, or gigabytes of information. This large amount ofinformation is difficult to interpret. To aid in the interpretation ofthe data, the data is commonly plotted and graphs and examined for peaksand patterns representing ions and ion fragments.

In accordance with the present invention, the data pair information issorted, by the computer processing means 15, into groups defined by arange of retention times into bins. The computer processing means 15 hasdata base means comprising mass and retention time information for knowncompounds. The computer processing means 15 initiates an ion auditprocess having at least one first pass which is described below. Thedata base of the computer processing means 15 may comprise stored datain the computer or data held in a data storage system or network.

The first pass of the ion audit process comprises the step of specifyingmatching criteria for data pair information present in a bin or aplurality of bins, potentially representing a compound, to form matchparameters. That is, the data pair information is examined for datapairs having the highest intensity representing a compound whichcompound is present in substantial quantity.

The computer processing means 15 identifies a group of data pairscorresponding to the match parameters forming a matched set. Next, thecomputer processing means compares the matched set to the data basemeans and identifying a compound corresponding to at least one of thematch parameters. The computer processing means 15 assigns anidentification to the data pairs of the matched set to form a scoredcompound. The computer processing means 15 subtracts the data pairs ofthe matched set from the bins to form one or more depleted bins.

This process is repeated with the data pair information held in the oneor more bins and depleted bins, until the ion audit process associatesall data pair information with compounds, fragments of compounds andreaction products of compounds, or the match criteria for remainingcompounds, fragments of compounds and reaction products of compounds donot correspond to information in the data base, or a user defined numberof repetitions.

Preferably, the ion audit process focuses on the dominant signals; theaudit clears such signals from the data pair information to allowidentification of the next dominant signal group. Thus, as the auditprogresses, the lower intensity signals are revealed with ever greaterclarity.

FIG. 2 a graphically depicts the binned data pairs by count andretention time. Only a single peak is observed. However, uponsubtracting the information of the data pairs for which a scoredcompound has been made, three smaller peaks become apparent. Thesesmaller peaks may comprise additional compounds.

Preferably, the ion audit process comprises specifying matching criteriafor data pair information present in a bin, depleted bin or a pluralityof bins or depleted bins, potentially representing one or more fragmentsof the scored compound, to form fragment match parameters. Next, a groupof data pairs corresponding to the fragment match parameters areidentified to form a group of data pairs forming a fragment associatedmatched set. The fragment associated matched set is subtracted from thebins and depleted bins. The further depleted bins have fewer data pairsallowing other groups of data pairs to be recognized with greaterclarity. The data pairs which represent fragments of the scored compoundare used to increase confidence in the compound identification.

Preferably, the ion audit comprises specifying matching criteria fordata pair information present in a bin or depleted bin or a plurality ofbins or depleted bins, potentially representing one or more precursorsof the scored compound, to form precursor match parameters. Next, agroup of data pairs corresponding to the precursor match parameters areidentified, to form a group of data pairs forming a precursor associatedmatched set. The precursor associated matched set is subtracted from thebins and depleted bins. The further depleted bins have fewer data pairsallowing other groups of data pairs to be recognized with greaterclarity. The data pairs which represent precursors of the scoredcompound are used to increase confidence in the compound identification.

Preferably, the ion audit comprises specifying matching criteria fordata pair information present in a bin or depleted bin or a plurality ofbins or depleted bins, potentially representing reaction products of thescored compound, to form reaction product match parameters. Next, datapairs which correspond to the reaction product match parameters areidentified to form a group of data pairs forming a reaction productassociated matched set. The reaction product associated matched set issubtracted from the bins and depleted bins. Again, the further depletedbins have fewer data pairs allowing other groups of data pairs to berecognized with greater clarity. The data pairs which represent reactionproducts of the scored compound are used to increase confidence in thecompound identification.

The apparatus can perform the ion audit in a single pass of all bins ora subset or a single bin. However, it is preferable to apply thesubtraction to a plurality of bins such that data pairs associated witha putative compound are removed and other details seen.

Preferably, the ion audit process includes a second pass, wherein for ascored compound, at least one selected from the group consisting ofanticipated fragments, anticipated precursors and anticipated reactionproducts thereof, are identified from the data base for the scoredcompound. Matching criteria for data pair information present in one ormore bins to form are specified defining second pass match parameters tosaid selected anticipated fragment, anticipated precursor andanticipated reaction product thereof. Data pairs which correspond tosaid second pass match parameters are identified to form a group of datapairs forming a second pass matched set. The second pass matched set issubtracted from said bins and depleted bins. A preferred second pass isdirected to the anticipated reaction products.

Thus, applying the process to all bins for a scored compound allows thedata pair information to be simplified, reduces the dominance of thelarger signals to allow lesser signals to be seen and identified. Theselesser signals may be obscured in bins not the initial focus of thescored compound but are revealed as signals associated with precursors,fragments and reaction products are removed.

Mass spectrometer 19 has a first mass selection chamber 41,fragmentation chamber 43 and a second mass detection chamber 45. Thefirst mass selection chamber 41 selects ions of a selected mass chargeratio and passes such ions to the fragmentation chamber 43. Thefragmentation chamber 43 can operate at low energy levels and highenergy levels. At low energy levels the compounds entering thefragmentation chamber 43 are not broken into fragments and are passed tothe second mass detection chamber 45 where such ions create a signalsent to the computer processing means 15. At high energy levels, thefragmentation chamber 43 causes the ions to form one or more fragmentsof the original compound. The fragments are passed to the second massdetection chamber 45 where such ions create a signal sent to the controlmeans.

Mass spectrometer 19 can switch from high to low energy mode under thecontrol of the computer processing means 15. Computer processing means15 preferably sets a time interval for cycling between the high and lowenergy levels smaller than the interval associated with the bins. Thus,mass spectrometers 19 with fragmentation chambers 43 are usedadvantageously with the computer processing means 15 to cycle betweenhigh and low energy such that fragments can be more readily beassociated with the parent compounds.

The apparatus 11 further comprises or is used with data output means 51in the form of a monitor for displaying the compounds identified in theion audit process. Those skilled in the art will recognize that the dataoutput means may comprise, in addition or in place of the monitor,printers, other video output devices, such as televisions, cathode raytubes, LCD screen, projectors and the like.

An embodiment of the present invention, directed to a method ofidentifying a compound, will be described with respect to the apparatus11. The chromatographic apparatus 17 receives a sample and separates thesample into compounds by retention time. The chromatographic apparatus17 produces a signal from which retention time can be determined. Themass spectrometer 19 receives one or more compounds from thechromatographic apparatus 17 and produces a signal corresponding to themass of the compound. And, in the event the compound forms one or morefragments, a signal corresponding to a fragment, and in the event thecompound reacts to form a reaction product, a signal corresponding tothe reaction product, and in the event the compound is present as aprecursor, a signal corresponding to the precursor.

Turning now to FIG. 3, which depicts a flow diagram of the method, themethod comprising an ion audit process having the steps of correlatingeach mass signal to a retention time signal to form a data pair and datapair information for a plurality of data pairs, storing the data pairinformation, and sorting the data pair information into groups definedby a range of retention times into bins. The method further comprisesthe step of specifying matching criteria for data pair informationpresent in a bin or a plurality of bins, potentially representing acompound, to form match parameters. And, the method comprises the stepof identifying a group of data pairs corresponding to the matchparameters, the data pairs corresponding to the match parameters forminga matched set. Next, the matched set are compared with data base meansand a compound is identified that corresponds to at least one of saidmatch parameters to assign an identification to the data pairs of thematched set to form an scored compound. The data pair information ofsaid matched set are subtracted from said bins to form one or moredepleted bins. The ion audit of the present invention repeats the stepsof specifying matching criteria through the step of subtracting datapair information from the bins and depleted, until all data pairinformation is associated with compounds, fragments of compounds andreaction products of compounds, or the match criteria for remainingcompounds, fragments of compounds and reaction products of compounds donot correspond to information in the data base, or a user defined numberof repetitions.

Preferably, the method comprises the step of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing one or more fragments of the scoredcompound, to form fragment match parameters. Next, a group of data pairscorresponding to the fragment match parameters are identified to form agroup of data pairs forming a fragment associated matched set. The datapair information of fragmented associated matched sets is subtractedfrom the bins and depleted bins. The data pairs which representfragments of the scored compound are used to increase confidence in thecompound identification. Removal of the data pair information allowsother features of the data to be seen. This is further illustrated as acontinuous process in FIG. 4.

Preferably, the method comprises the step of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing one or more precursors of the scoredcompound, to form precursor match parameters. Next, a group of datapairs corresponding to said precursor match parameters are identified toform a group of data pairs forming a precursor associated matched set.The data pair information of the precursor associated matched set issubtracted from the bins and depleted bins. The data pairs whichrepresent precursors of the scored compound are used to increaseconfidence in the compound identification. Removal of the data pairinformation allows other features of the data to be seen.

Preferably, the method comprises the steps of specifying matchingcriteria for data pair information present in a bin or a plurality ofbins, potentially representing a reaction products of the scoredcompound, to form reaction product match parameters. Data pairs whichcorrespond to said reaction product match parameters are identified toform a group of data pairs forming a reaction product associated matchedset. The data pair information of the reaction product associatedmatched set is subtracted from the bins and depleted bins. The datapairs which represent reaction products of the scored compound are usedto increase confidence in the compound identification. Removal of thedata pair information allows other features of the data to be seen.

The method of the present invention can be performed in a single firstpass or in multiple passes. FIGS. 4 a and 4 b depict a continuousprocess performed in a plurality of passes. Preferably, the ion auditprocess includes a second pass, wherein for a scored compound at leastone selected from the group consisting of anticipated fragments,anticipated precursors and anticipated reaction products thereof ischosen for a second pass. The second pass follows a first pass or afirst series of passes in which the specifying criteria remainssubstantially unchanged.

Returning now to FIG. 1, preferred method is performed by computerprocessing means 15 by means of software. An outline of the softwareprogram is set forth in the following sixteen pages. Softwareprogrammers are able to readily develop source code directed to theinvention described herein and in reference to the outline.

Thus, the present invention has been described with respect to preferredembodiments with the understanding that those skilled in the art will beable to make alterations and modifications to the invention. Therefore,the invention should not be limited to the precise details set forthherein but should encompass the subject matter of the following claimsand their equivalents.

The invention claimed is:
 1. A method for identifying compounds in asample comprising: processing said sample using a chromatographicseparation apparatus and a mass spectrometer to obtain sample dataincluding precursor ion information for precursor ions and product ioninformation for product ions derived from compounds in the sample, saidprecursor ion information for each of said precursor ions including amass and a retention time for said each precursor ion, said product ioninformation for each of the product ions including a mass and aretention time for said each product ion; determining a list of rawmatches between masses and retention times of precursor and product ioninformation of said sample data and masses and retention times ofprecursor and product ion information associated with a plurality ofknown compounds in a database; identifying a first of the plurality ofknown compounds in the sample by matching masses and retention times ofa first set of precursor ion information and product ion information ofthe first compound from the database to masses and retention times of asecond set of precursor ion information and product ion information fromthe list of raw matches, said first set including precursor informationfor precursor ions and associated product ions for the first compound,said first compound having a highest abundance of any identified one ofthe plurality of known compounds in the sample; removing from the listof raw matches said second set of precursor ion information and production information thereby obtaining a revised list of raw matches; andrepeating said identifying and said removing using the revised list ofraw matches to identify a second of the plurality of known compounds. 2.The method of claim 1, wherein said first compound is identified ashaving the highest abundance using ion intensities associated with ionsof the second set.
 3. The method of claim 2, wherein the highestabundance is determined by summing ion intensities of product ionshaving their product ion information included in the second set.
 4. Themethod of claim 1, wherein said compounds in the sample are proteins andthe database includes precursor and associated product ion informationfor a plurality of known proteins.
 5. The method of claim 1, wherein thefirst set includes precursor information for at least two precursors andat least six product ions associated with said at least two precursors.6. The method of claim 1, wherein said determining, said identifying,said removing and said repeating are performed in a first pass of anion-audit process, said first pass identifying a portion of saidplurality of known compounds as being included in said sample, whereineach compound identified in said portion has a plurality of precursorions that have been validated by a plurality of product ions associatedwith said plurality of precursor ions.
 7. The method of claim 6, whereinsaid ion-audit process includes performing a second pass, said secondpass comprising processing including assigning an unvalidated precursorto one the identified compounds of the portion wherein the unvalidatedprecursor is not associated with any product ions.
 8. The method ofclaim 1, wherein each mass included in the sample data is correlatedwith a retention time and said each mass corresponds to a mass of any ofa compound, a product ion formed from a compound, a precursor ion formedfrom a compound, and a reaction product of a compound.
 9. The method ofclaim 8, wherein the reaction product comprises at least one of thegroup consisting of oxidized, methylated, phosphorylated andglycosylated fragments and precursors.
 10. A computer readable mediumcomprising code stored thereon for identifying compounds in a sample,the computer readable medium comprising code stored thereon forperforming processing including: correlating each mass signal receivedfrom a mass spectrometer to a retention time signal received from achromatographic apparatus to form a data pair and data pair informationfor a plurality of data pairs; sorting said data pair information intogroups defined by a range of retention times into bins; specifyingmatching criteria for data pair information present in a bin or aplurality of bins, potentially representing a compound, to form matchparameters and identifying a group of data pairs corresponding to saidmatch parameters, said data pairs corresponding to said match parametersforming a matched set; identifying a first compound corresponding to atleast one of the match parameters by comparing said matched set to adatabase including information on the first compound; assigning anidentification to said data pairs of the matched set to form a scoredcompound; removing said data pairs of the matched set from the bins toform one or more depleted bins; and repeating said specifying, saididentifying, said assigning and said removing with data pair informationincluded in said depleted bins.
 11. The computer readable medium ofclaim 10, wherein each mass signal received from the mass spectrometercorresponds to the mass of any of a compound, a product ion formed froma compound, a precursor ion formed from a compound, and a reactionproduct of a compound.
 12. A system, for identifying compounds in asample comprising: a chromatographic separation apparatus that separatecompounds in the sample by retention time; a mass spectrometer thatreceives one or more compounds separated by the chromatographicseparation apparatus and generates sample data including precursor ioninformation for precursor ions and product ion information for productions derived from compounds in the sample, said precursor ioninformation for each of said precursor ions including a mass and aretention time for said each precursor ion, said product ion informationfor each of the product ions including a mass and a retention time forsaid each product ion; and a computer readable medium comprising codestored thereon for identifying compounds in a sample, the computerreadable medium comprising code stored thereon for: determining a listof raw matches between masses and retention times of precursor andproduct ion information of said sample data and masses and retentiontimes of precursor and product ion information associated with aplurality of known compounds in a database; identifying a first of theplurality of known compounds in the sample by matching masses andretention times of a first set of precursor ion information and production information of the first compound from the database to masses andretention times of a second set of precursor ion information and production information from the list of raw matches, said first set includingprecursor information for precursor ions and associated product ions forthe first compound, said first compound having a highest abundance ofany identified one of the plurality of known compounds in the sample;removing from the list of raw matches said second set of precursor ioninformation and product ion information thereby obtaining a revised listof raw matches; and repeating said identifying and said removing usingthe revised list of raw matches to identify a second of the plurality ofknown compounds.
 13. The system of claim 12, wherein said first compoundis identified as having the highest abundance using ion intensitiesassociated with ions of the second set.
 14. The system of claim 13,wherein the highest abundance is determined by summing ion intensitiesof product ions having their product ion information included in thesecond set.
 15. The system of claim 12, wherein said compounds in thesample are proteins and the database includes precursor and associatedproduct ion information for a plurality of known proteins.
 16. Thesystem of claim 12, wherein the first set includes precursor informationfor at least two precursors and at least six product ions associatedwith said at least two precursors.
 17. The system of claim 12, whereinsaid determining, said identifying, said removing and said repeating areperformed in a first pass of an ion-audit process, said first passidentifying a portion of said plurality of known compounds as beingincluded in said sample, wherein each compound identified in saidportion has a plurality of precursor ions that have been validated by aplurality of product ions associated with said plurality of precursorions.
 18. The system of claim 17, wherein said ion-audit processincludes performing a second pass, said second pass comprisingprocessing including assigning an unvalidated precursor to one theidentified compounds of the portion wherein the unvalidated precursor isnot associated with any product ions.
 19. The system of claim 12,wherein each mass included in the sample data is correlated with aretention time and said each mass corresponds to a mass of any of acompound, a product ion formed from a compound, a precursor ion formedfrom a compound, and a reaction product of a compound.
 20. The system ofclaim 19, wherein the reaction product comprises at least one of thegroup consisting of oxidized, methylated, phosphorylated andglycosylated fragments and precursors.